Metabolic engineering of Escherichia coli to enhance protein production by coupling ShCAST-based optimized transposon system and CRISPR interference

CRISPR-Cas9 induces double-strand break (DSB) for gene editing, but integration efficiency is low in some E. coli strains such as BL21(DE3). CRISPR-associated transposon from cyanobacterium Scytonema hofmanni (ShCAST) is a novel system comprising transposase and Cas12k for RNA-guided, DSB-free DNA transposition. However, ShCAST was developed in the specialized E. coli strain and its application is limited by off-target effect and plasmid DNA co-integration. To overcome the drawbacks of ShCAST, we developed a ShCAST-based Optimized Transposition (SHOT) methodology by elevating Cas12k expression with an independent strong promoter, lowering the editing temperature and colony re-streaking. SHOT enhanced on-target integration into sites difficult to edit by ShCAST without detectable off-target effects, and eliminated plasmid co-integration in BL21(DE3). SHOT enabled ≈97% integration of large DNA (≥14.5 kb) and multiplexed integration into difficult-to-edit loci, hence expediting generation of stable strains. We further leveraged SHOT to integrate a 10.3 kb CRISPR interference module into BL21(DE3) for multiplexed suppression of acid-producing genes, hence attenuating acid byproduct formation and increasing recombinant protein production. SHOT also facilitated DSB-free engineering of other difficult-to-edit E. coli and bacterial strains. Collectively, SHOT overcomes the shortcomings of CRISPR-Cas9 and ShCAST and facilitates genome engineering of difficult-to-edit E. coli strains.